Name: Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)
Uploaded: 2023-05-05T12:10:00
Duration: 59 s
Description: Watch this Scientific Journal Video about Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation ChIP at JoVE.com

collection of f1 generation caenorhabditis elegans for chromatin immunoprecipitation (chip)

Epigenetische overerving en ChIP bij Caenorhabditis elegans

Epigenetic inheritance allows the transmission of gene regulatory information across generations and can therefore allow the environment or experiences of parents to affect their progeny. In C. elegans, germline gene silencing initiated by exogenous double-stranded RNA (dsRNA) can be inherited for multiple generations in progeny not exposed to the original trigger1,2,3,4. This process, termed RNA interference (RNAi) inheritance, is one of several related epigenetic silencing phenomena in C. elegans, including piRNA-initiated multigenerational silencing2,5, paramutation/RNAe (RNA-induced epigenetic silencing)6,7,8, and multicopy array-initiated silencing9, which have overlapping but distinct requirements for small RNA and chromatin regulation machinery. In C. elegans exogenous RNAi, dsRNA is processed into small interfering RNAs (siRNAs) that act in a complex with primary Argonaute proteins to recognize their target mRNA. This targeting leads to the amplification of secondary siRNAs that associate with secondary Argonautes to silence target mRNA through both cytoplasmic and nuclear silencing pathways. For germline-expressed RNAi targets, the nuclear secondary Argonaute HRDE-1 and additional nuclear RNAi factors target nascent transcripts, resulting in transcriptional repression and recruitment of histone methyltransferases to deposit repressive chromatin marks, including H3K9me310. Histone H3K9me3 promotes the establishment of heritable silencing of germline-expressed green fluorescent protein (GFP) transgenes by RNAi inheritance11,12.
The goal of this protocol is to use a transgene expressing a GFP-histone fusion protein in the germline as a reporter for RNAi inheritance and to assay changes in histone modifications at the reporter transgene locus using chromatin immunoprecipitation and quantitative polymerase chain reaction (ChIP-qPCR). This protocol describes a standardized plate-based RNAi feeding approach to initiate reporter silencing. It also provides a detailed timeline for passaging animals between generations by isolating in utero embryos from gravid adults by alkaline hypochlorite treatment (&#39;bleaching&#39;). Methods and representative data for monitoring the frequency of GFP silencing in a subset of the population by fluorescence microscopy and for histone H3K9me3 ChIP-qPCR are also described. Reporter-based RNAi inheritance assays provide a highly tractable system to functionally dissect the roles of genetic and environmental factors in epigenetic regulation13,14, and genetic screens using such reporters have identified both genes that are required for2,3,15 and genes that negatively regulate16,17 the duration of transgenerational epigenetic inheritance.

Auteur in de schijnwerpers: RNAi-overerving en ChIP in C. elegans 

Analyse van transgenerationele epigenetische overerving in C. elegans met behulp van een fluorescerende reporter en chromatine-immunoprecipitatie (ChIP)

Research in our lab examines how chromatin-associated proteins, histone modifications, and small RNA pathway factors work together in the context of development and transgenerational epigenetic inheritance. The RNA inheritance assay, using a GFP reporter, has become a very powerful tool. This protocol describes how to standardize the preparation, scoring, and passaging of worms, and the preparation of ChIP samples, which can help to generate reproducible results.One of the experimental challenges when studying germline-expressed transgenes is isolating the effects in the animal's germline. We're currently using whole animals for ChIP, but in the future we would like to develop assays to examine chromatin, specifically in the germline. Our laboratory is interested in histone methylation and proteins that recognize these marks.Going forward, we would like to investigate the interplay between these chromatin readers and small RNA pathways in the context of RNAi inheritance at transgenes and the regulation of endogenous sequences.

Watch this Scientific Journal Video about Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation ChIP at JoVE.com

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)  

Verzameling van F1-generatie Caenorhabditis elegans voor chromatine-immunoprecipitatie (ChIP)

Begin met het plateren van ongeveer 3.500 C.elgans F1-generatie embryo's op 14 platen en laat ze drie dagen bij 21 graden Celsius uitgroeien tot het jongvolwassen stadium. Was en verzamel de C.elgans in buizen van 1.5 milliliter met behulp van PBS/TX. Centrifugeer gedurende twee minuten bij 1000 G, zuig op en trek de wormen van één stam in één buis.Herhaal drie wasbeurten met PBS/TX. Zuig vervolgens het supernatans op en laat een milliliter in de buis achter en keer om om te mengen. Nadat u drie keer het aantal C.elgans-wormen in drie microliter hebt geteld, berekent u de concentratie wormen en brengt u een volume over dat overeenkomt met 3000 wormen naar een nieuwe buis voor formaldehyde-verknoping.

Research

JoVE Journal

Genetics

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates  

Bleken en plateren van Caenorhabditis elegans P0 Generation Embryo's op RNAi-NGM-platen

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression  

Passeren en scoren van de Caenorhabditis elegans P0-generatie voor kiembaan-GFP-expressie

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP  

Formaldehyde-verknoping van de F1-generatie van Caenorhabditis elegans voor ChIP

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation  

Sonicatie van formaldehyde-verknoopte Caenorhabditis elegans en chromatine-immunoprecipitatie